Omega-3 (or ω-3) polyunsaturated fatty acids, in particular docosahexaenoic acid (DHA), are especially important during prenatal brain development and maintenance of human brain function. Compared with saturated fatty acid, polyunsaturated fatty acid with multiple double bonds within molecule, in particular DHA, causes carbon-carbon chains to become more curved. The more kinked the fatty acid is, the more space it will take up, when it is built into cell membrane phospholipids, in order to keep neuronal membrane functions. This is the main reason that why the brain requires a large amount of nutritionally essential polyunsaturated fatty acids, especially DHA, because DHA and DHA-containing molecular species of phospholipids may contribute to important brain functions including signal transduction and information processing [Akbar et al., Docosahexaenoic acid: a positive modulator of Akt signaling in neuronal survival. Proc. Natl. Acad. Sci. U.S.A. 102: 10858 (2005)]. Alteration of neuronal membrane DHA-containing phospholipid species can not only influence crucial intracellular and intercellular signaling but also alter many membrane physical properties such as fluidity, phase transition temperature and bilayer thickness. The deficiency of DHA markedly affects neurotransmission, membrane-bound enzyme and ion channel activities leading to brain aging, Alzheimer's disease, Parkinson's disease, schizophrenia and peroxisomal disorders. For example, a study indicated that the concentration of DHA in patients with Alzheimer's disease is significantly decreased [Conquer, et al., Fatty acid analysis of blood plasma of patients with Alzheimer's disease, other type of dementia, and cognitive impairment, Lipids, 35:1305 (2000)]. The studies of Garcia et al. [Garcia et al., Effect of docosahexaenoic acid on the synthesis of phosphatidylserine in rat brain in microsomes and C6 gliome cells. J. Neurochem. 70: 24 (1998); Kim et al., Inhibition of neuronal apoptosis by docosahexaenoic acid (22:6n-3): Role of phosphatidylserine in anti-apoptotic effect. J. Biol. Chem. 275:35215 (2000)] found out the new role of DHA and phosphatidylserine in neuronal apoptosis, indicating that exogenous DHA may enhance phosphatidylserine accumulation in apoptotic Neuro-2A cells leading to the protection of neuronal cells from apoptotic death. The studies strongly suggest that one of supporting roles for anti-apoptosis of neurons is supplying DHA to the brain.
Because the human body cannot synthesize ω-3 polyunsaturated fatty acids, in particular DHA, exogenous introduction of DHA to human has been applied. There are a few products available for use as brain nutrients, such as fish oils (DHA-containing neutral lipids) and similar products.
Although these products contain DHA and other omega-3 polyunsaturated fatty acids, experiments have demonstrated that only a very small amount of DHA can be found in the brain after administering a large amount of these products. But an early study showed that DHA-containing lysophospholipid in albumin, rather than the forms of free DHA and other esterified DHA, is preferred in the uptake of DHA in the brain of young rats when an in vitro model of blood-brain barrier is used [Thies et al., Unsaturated fatty acids esterified in 2-acyl-1-lyso-phosphatidylcholine bound to albumin are more efficiently taken up by the young rat brain than unesterified form. J. Neurochem. 59: 1110 (1992)].
Interestingly, a study reported that dietary phospholipid with DHA-containing molecular species as supplementation is much more efficient than soybean phospholipid for ensuring a normal level DHA in the brain during the period of brain development in rats [Bourre and Dumont, Neurosci. Lett., 335:129 (2002)] because DHA species are absent in the latter. The result suggests that DHA-containing phospholipid species are effective forms to be used as DHA carriers to brain.
Phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) as well as lysophosphatidylcholine (Lyso PC), lysophosphatidylserine (Lyso PS) and lysophosphatidyl-ethanolamine (Lyso PE) are naturally occurring phospholipid classes, existing in mixture forms of the molecular species. The structural diversity of the molecular species of phospholipids has been described in detail [Chen, Lipids, 32, 85 (1997); Chen et, al. Biomed. Mass Spectrom. 21, 655 (1992)]. Biochemical and biophysical functions of phospholipids are well documented and appear to be determined by the fatty acid composition of the lipids.
Ether phospholipids are usually found in animal tissues and human cells as minor components, existing together with molecular species of diacyl phospholipids carrying the same polar head group. It is well known that there are two predominant types of ether bonds in the phospholipid. One form is represented by the plasmalogens (with 1-alk-1′-enyl fatty chain linked to the sn-1 position of the glycerol backbone), which is the most abundant subclass of phospholipids in most tissues. The other form is alkyl phospholipids that contain 1-O-alkyl fatty chain(s) linked to the sn-1 position of the glycerol backbone. Although mixtures of phospholipids and ether phospholipids have been found in animals and humans [Diagne, et. al., Studies on ether phospholipids, Biochim. Biophys. Acta, 793, 221 (1984)], little has been described regarding the presence of phospholipid species mixtures containing 1-O-alkyl-2-DHA molecules, as well as DHA-containing lysophospholipids in aquatic animals.
Investigation of PC metabolism in human [Galli et al., Prolonged retention of doubly labeled phosphatidylcholine in human plasma and erythrocytes after oral administration. Lipids, 27: (1992)] indicated that a major portion of PC species can be found as intact molecules in plasma after oral administration of labeled phospholipid species. The result is supported by an animal experiment, suggesting that more than 80% of PC, which is recovered from the intestinal lymph of rats, is still intact after oral administration of phospholipids [Ikeda et al., Absorption and transport of base moieties of phosphatidylcholine and phosphatidylethanolamine in rats, Biochim. Biophys. Acta, 921; 245 (1987)].
However, fatty acid chains of phospholipids can be further hydrolyzed by lecithin-cholesterol acyltransferase in plasma after molecular species are incorporated into high density lipoprotein, and followed by phospholipase A1 and phospholipase A2 hydrolyses in the liver. Recently, several papers reported the possibility of transporting high density lipoprotein across the blood-brain barrier using an in vitro model [Balazs et al., Uptake and transport of high-density lipoprotein (HDL) and HDL-associated α-tocopherol by an in vitro blood-brain barrier model. J. Neurochem. 89: 939 (2004)] and the importance of endothelial lipase in the metabolism of high density lipoprotein associated phospholipids at the blood-brain barrier [Ma et al., Endothelial lipase is a major genetic determinant for high-density lipoprotein concentration, structure, and metabolism. Proc. Natl. Acad. Sci. U.S.A. 100: 2748 (2003)], demonstrating that endothelial lipase (i) exhibits primarily the activity of phospholipase A1 leading to the hydrolysis of sn-1 fatty acid chains from the molecular species of high density lipoprotein phospholipids, and (ii) is inactive in the hydrolysis of ether phospholipid molecular species [Gauster et al., Endothelial lipase release saturated and unsaturated fatty acid of high density lipoprotein phosphatidylcholine. J. Lipid Res. 46: 1517 (2005)].
An early study also showed that after the administration of lysophospholipids, these lipid species do not enhance fatty acid chain retention in mucosa and may survive from the hydrolysis of lecithin-cholesterol-acyltransferase in plasma, as well as other phospholipases in the liver [Viola et al., Absorption and distribution of arachidonate in rats receiving lysophospholipids by oral route, J. Lipid Res. 34, 1843 (1993)].
Based on these published results, it is clear to see that ether phospholipid species, such as 1-O-alkyl-2-acyl molecular species, and lysophospholipid molecular species are more stable in vivo lipid metabolism, compared with related acyl species. Because ether phospholipid and lysophospholipid species can be survived from blocking due to the hydrolysis of phospholipase A1 and phospholipase A1-like enzymes in vivo metabolism [Shamburek et al., Disappearance of two major phosphatidylcholine from plasma is predominantly via LCAT and hepatic lipase, Am J. Physiol. 271: E1073 (1996); Scagnelli, Plasma 1-palmitoyl-2-linoleoyl phosphatidylcholine. Evidence for extensive phospholipase A1 hydrolysis and hepatic metabolism of the products, J. Biol. Chem. 266: 18002 (1991)], DHA-containing ether phospholipid species and DHA-containing lysophospholipid species can be delivered smoothly into the brain as carriers of DHA, resulting in the uptake of free DHA after brain phospholipase hydrolyses of the phospholipids species [Ross et al., Characterization of a novel phospholipase A2 activity in human brain. J. Neurochem. 64, 2213 (1995)].
Interest is focused on the preparation of phospholipid species containing 1-O-alkyl-2-DHA molecules and DHA-containing lysophospholipid species. However, obtaining molecular species mixtures containing enriched DHA-containing ether phospholipids and -lysophospholipids species from aquatic animals is poorly understood [Chapelle, Plasmalogen and O-alkyl glycerophospholipids in aquatic animals, Comp. Biochem. Physiol. 88, 1 (1987)]. Additionally, the chemical synthesis of these phospholipids species is both difficult and expensive, and thus a large-scale preparation of the molecular species for use as potential brain health supplementation is not available. Thus there is a need for a method of a large-scale preparation of DHA-containing phospholipids and -lysophospholipids species for use as potential brain health supplementations.